Many vaccines including influenza vaccine for the treatment of humans and animals consist of one or more virus strains which have been replicated in embryonated hens' eggs. These viruses are isolated from the allantoic fluid of infected hens' eggs and their antigens are used in vaccines as intact virus particles, as virus particles disintegrated by detergents and/or solvents, as chemically or physically inactivated viruses, or as isolated, defined virus proteins as in subunit vaccines. The viruses are often inactivated by processes known to the person skilled in the art. The replication of live attenuated viruses, which are tested in experimental vaccines, is also carried out in embryonated hens' eggs.
The use of embryonated hens' eggs for vaccine production is time-, labor- and cost-intensive. The eggs, from healthy flocks of hens monitored by veterinarians, have to be incubated before infection, customarily for 12 days. Before infection, the eggs have to be selected with respect to living embryos, as only these eggs are suitable for virus replication. After infection the eggs are again incubated, customarily for 2 to 3 days. The embryos still alive at this time are killed by subjecting them to a cold environment, and the allantoic fluid is then obtained from the individual eggs by aspiration. By means of laborious purification processes, substances from the hen's egg that lead to undesired side effects of the vaccine are separated from the viruses, and the viruses are concentrated. As eggs are not sterile (pathogen-free), it is additionally necessary to remove and/or to inactivate pyrogens and all pathogens that are possibly present.
Viruses of other vaccines, such as, for example, rabies viruses, mumps, measles, rubella, polio viruses, tick bourne encephalits viruses such as Frühsommer-Meningo Ecephalitis (FSME) virus can be replicated in cell cultures. As cell cultures originating from tested cell banks are pathogen-free and, in contrast to hens' eggs, are defined virus replication systems that (theoretically) are available in almost unlimited amounts, they make possible economical virus replication under certain circumstances even in the case of influenza viruses. Moreover, the isolation and replication of influenza viruses in eggs leads to a selection of certain phenotypes, of which the majority differ from the clinical isolate. In contrast to this is the isolation and replication of the viruses in cell culture, in which no passage-dependent selection occurs (Oxford, J. S. et al., J. Gen. Virology 72(1991), 185-189; Robertson, J. S. et al., J. Gen. Virology 74 (1993) 2047-2051). For an effective vaccine, therefore, virus replication in cell culture is preferred.
It is known that influenza viruses can be replicated in cell cultures. Beside hens' embryo cells and hamster cells (BHK21-F and HKCC), MDBK and MDCK cells have been described as suitable cells for the in-vitro replication of influenza viruses (Kilbourne, E. D., in: Influenza, pages 89-110, Plenum Medical Book Company-New York and London, 1987). A prerequisite for a successful infection is the addition of proteases to the infection medium, preferably trypsin or similar serine proteases, as these proteases extracellularly cleave the precursor protein of hemagglutinin [HA0] into active hemagglutinin [HA1 and HA2]. Only cleaved hemagglutinin leads to the adsorption of the influenza viruses on cells with subsequent virus assimilation into the cells (Tobita, K. et al., Med. Microbiol. Immunol., 162 (1975), 9-14; Lazarowitz, S. G. & Choppin, P. W., Virology, 68 (1975) 440-454; Klenk, H.-D. et al., Virology 68 (1975) 426-439) and thus to a further replication cycle of the virus in the cell culture.
U.S. Pat. No. RE 33,164 (from U.S. Pat. No. 4,500,513), which is wholly incorporated by reference herein, described the replication of influenza viruses in cell cultures of adherently grown CLDK cells (or, “aCLDK cells”). The constraining requirement of growing these cells adherently places a limitation on the yield of cells that can be grown and also consequently places a limitation on the yield of virus that can be harvested for formulation in a vaccine.
Moreover, growing virus in adherent (or, substrate-dependent) cells requires steps not necessary when the cells can be grown in suspension. After cell proliferation, the nutrient medium is removed and fresh nutrient medium is added to the cells with infection of the cells with influenza viruses taking place simultaneously or shortly thereafter. A given time after the infection, protease (e.g. trypsin) is added in order to obtain an optimum virus replication. The viruses are harvested, purified and processed to give inactivated or attenuated vaccine.
Economical influenza virus replication as a prerequisite for vaccine production cannot be accomplished, however, using the methodology described in U.S. RE 33,164, as the change of media, the subsequent infection as well as the addition of trypsin, which is carried out later, necessitates opening the individual cell culture vessels several times and is thus very labor-intensive. Furthermore, the danger of contamination of the cell culture by undesirable micro-organisms and viruses increases with each manipulation of the culture vessels. Yet another disadvantage with this system is that serum (including without limitation fetal calf serum, fetal bovine serum (fbs), newborn calf serum or bovine serum) is necessary for the growth of the cells. Serum contains trypsin inhibitors that interfere with viral yield.
A more cost-effective alternative is cell proliferation in systems where the cells do not need to be grown adherently to the culture container or on the surface of micro carriers. U.S. Pat. No. 6,656,720, which is wholly incorporated by reference herein, provides an example of one such method wherein MDCK cells that are grown in suspension are infected with influenza virus. However, additional cell lines and methodologies are needed that provide alternative means of growing viruses to increase efficiencies and reduce overall costs.
Hence, there is a need for additional cell lines that can be cultured in medium that is free of animal-derived components (e.g., serum-free medium or animal protein-free medium) to reduce the risk associated with use of animal by-products (e.g., bovine serum) and to eliminate the expense of such animal by-products. Furthermore, there is also a general need to eliminate the necessity of substrate-dependent growth (e.g., T-flask, roller bottle or micro carriers) and to have suspension cultures instead. Suspension cultures have numerous advantages over substrate-dependent growth including cost savings, higher cell densities and greater virus yields.